1. Field of the Invention
The present invention relates to a gene encoding a lectin specifically binding to sialoglycoconjugates, which is originated from the mushroom Polyporus squamosus, an active lectin protein obtained by expressing the said gene in a heterologous host, a recombinant transformant producing the said protein, and a method for measuring the biological material including glycoproteins, glycopeptides, glycolipids, sugar precursors, or oligosaccharides containing sialic acid moieties.
2. Description of the Related Art
Sialic acid (Neuraminic acid; Neu5Ac) is the alpha-ketoaldonic acid containing 9 carbons, which is a sugar found in the non-reduced end of a glycoconjugate such as glycoprotein, glycolipid, and oligosaccharide. Again, sialic acid is a sugar existing at the end of a glycoconjugate. Up to date, approximately 80 kinds of glycoconjugates bound with sialic acid have been identified in the natural world. Sialic acid plays an important role in biological phenomenon in cells, for example cell/cell interaction, functioning as a precursor determining the intracellular signal, and stabilization of a glycoprotein, etc. In particular, endogenous sialic acid is known as one of the glycans that is recognized first after the infection of a pathogen (Sasisekharan and Myette, 2003, Am. Sci. 91, 432; Vimr and Lichtensteiger, 2002, Trends Microbiol. 10, 254). Sialic acid on the cell surface is also known as a component forming a poly-oligosaccharide in the form of a capsule for cell protection or involved in immune privilege even in the pathogen itself (Vimr et al., 2004, Microbiol. Mol. Biol. Rev. 68, 132). The chemical structure of sialic acid is very complicated, which is composed of the carboxyl group connected to anomeric site of the second carbon and the glycerol structure found in deoxy of the third carbon and the sixth carbon. In the sialoglycoconjugate structure, the sialic acid is linked to other sugars via alpha(2,3)-, alpha(2,6)-, alpha(2,8)-, and alpha(2,9)-linkage. In addition the hydroxyl residue of sialic acid sugar is substituted with O-acetyl, O-phosphate, O-sulfate, O-methyl, and N-glycosyl group, resulting in producing a more complicated chemical structure such as transformants with N-glycolylneuraminic acid (NeuGc). Because of such a complicated chemical structure, it is very difficult to measure glycoconjugates containing sialic acid accurately. Due to the advanced new analysis techniques based on mass spectrometer and nuclear magnetic resonance spectrometer, etc, the analysis of a glycan structure containing sialic acid is more accurately achieved than before. However, there is still a problem to solve such as the lack of the amount of a biological sample and the complexity in the preparation of a sample for the analysis (Cohen and Varki, 2010, OMICS 14, 455; Alley et al. 2013, Chem. Rev. 113, 2668).
To measure the complicated structure of a sialic acid conjugated glycans, lectins have been applied to detect glycan structures (Vanderschaeghe et al., 2010, Biol. Chem. 391, 149; Hsu and Mahal, 2009, Curr. Opin. Chem. Biol. 13, 427). Lectins are multivalent carbohydrate binding proteins that display neither catalytic activity nor antibody like characteristics. Lectin, found in a wide range of living things from microorganisms to higher animals, is one of the proteins that can bind to sugar chains, which is involved in various life phenomena including protein quality control, host-pathogen interaction, cell-cell communication, inflammation, immune response, cancer progression, and development, by binding specifically to glycans (Lam and Ng, 2011. Appl. Microbiol. Biotechnol. 89, 45). Lectin was first isolated from Ricinus communis by the Russian scientist Sillmark in the late 19th century. Since then, 40 kinds of lectins including Concanavlain A (ConA) have been identified, studied, and used in various fields. Those lectins identified so far are extracted and purified from various natural bioresources, which are used to detect sialic acid (Neu5Ac, Neuraminic acid), galactose (Gal), N-acetylglucosamine (N-GlcNAc), fucose (Fuc), mannose (Man), and glucose (Glc) (Lehmann et al., 2006, Cell. Mol. Life Sci. 63, 1331; Singh et al., 2010 Crit. Rev. Biotechnol. 30, 99; Kajiwara et al., 2010, Microbes Environ. 25, 152).
However, due to the variety of structures of glycoconjugates having alpha and beta linkages and the variety of possible monomer combinations in glycoconjugates or side-chain modifications, hundreds or thousands of different glycan structures are possible theoretically. However the limited number of identified lectins are available for detection of a variety of glycan structures. In particular, Maackia amurensis (MAA), Sambucus nigra (SNA), and Limulus polyphemus lectin can be used for detection of sialic acid (Neu5Ac) containing glycan structures but the lectin could not accurately distinguish α2,3-, α2,6-, and α2,8-linkages, the chemical specificity of sialic acid sugar chain, due to isolectin contamination in the extracted lectin proteins. Besides, the lectin sometimes recognizes specifically Gal and GalNAc binding to sialic acid, leading to non-specific signal because of the non-specific binding. Therefore, it has been requested to develop a novel sialic acid specific lectin with elimination of isolectin interference.
The lectin of the present invention was isolated and purified from Polyporus squamosus fruiting body by using β-galactosyl-Synsorb column and DEAE-Sephacel ion exchange column, which was approximately 28 kDa and bound specifically to Neu5Ac-α(2,6)-Gal-β(1,4)-Glc/GlcNAc sugar chain (Mo et al., 2000, J. Biol. Chem. 275, 10623). The cDNA sequence obtained from Polyporus squamosus by RACE (rapid amplification of cDNA ends) using the degenerated primers constructed based on the partial amino acid sequence of the corresponding lectin was the protein encoding two isolectins PSL1a and PSL1b having 858 bp and 876 bp long sequences respectively. These are presumed to be the proteins in the sizes of 31.04 kDa and 31.998 kDa theoretically (Tateno et al. 2004, Biochem. J. 382, 667). The nucleotide sequence encoding PSL1a lectin is expressed in E. coli and isolated/purified as an active recombinant protein, whose characteristics have been disclosed. Recently the three-dimensional protein structure of PSL1a lectin was identified as the human-type influenza receptor Neu5Acα(2,6)Galβ(1,4)GlcNAc (Tateno et al. 2004, Biochem. J. 382, 667; Kadirvelraj et al., 2011, Glycobiology 21, 973). However, the characteristics of the nucleotide sequence encoding PSL1b lectin cannot be identified yet since it is generated as an inactive protein in E. coli (Tateno et al. 2004, Biochem. J. 382, 667).
The present inventors tried to obtain Polyporus squamosus-derived isolectin PSL1b from a heterologous host as an active recombinant protein that binds specifically to sialic acid. As a result, the inventors chemically synthesized the polynucleotide encoding isolectin PSL1b lectin protein and at last obtained a novel active recombinant lectin protein from the heterologous host system such as E. coli and yeast. The present inventors further confirmed that the constructed novel recombinant lectin protein can be used to measure the proteins having different sugar chain structures such as glycoproteins, glycolipids, and oligosaccharides containing sialic acid moieties, to monitor microorganisms, and to explain various life phenomena mediated by glycoconjugates, leading to the completion of the invention.